No approved medicament yet exists for treating the cause of Alzheimer's dementia (AD). Deposits of the so-called beta-amyloid peptide (Aβ) in plaques are typically found post mortem in the brains of AD patients. Various forms of Aβ, for example fibrils, have therefore long been blamed for the onset and progression of AD.
For the past few years, small Aβ aggregates (Aβ oligomers) in particular have been blamed as the main culprit for the onset and progression of AD. A reduction or complete elimination of Aβ oligomers would thus appear to be the most important criterion for curing or slowing AD.
Aβ monomers are constantly being produced in our body and are presumably not toxic per se. There is speculation as to whether Aβ monomers agglomerate randomly depending on their concentration, which ultimately results from the rate at which they are formed and broken down in the body, and thus are increasingly more likely to form Aβ oligomers spontaneously as a person gets older. Once formed, Aβ oligomers could then multiply through a prion-like mechanism and ultimately lead to the disease.
Based on these considerations, causal treatment should aim at completely destroying toxic Aβ oligomers and/or hindering the prion-like multiplication thereof. One important point here is the fact that any active ingredient has to be tested in an animal model and in clinical studies. These are very time-consuming and costly. A rapid, reliable and quantitative in vitro analysis, which pre-selects the most effective active ingredients against Aβ oligomers, would be of great advantage.
Some years ago, a D-enantiomeric peptide having the name D3 was identified by a mirror image phage display selection against predominantly monomeric Aβ(1-42), with the plan of stabilizing it by way of binding and preventing the conversion thereof into toxic Aβ aggregates. According to what is presently known, D3 destroys the particularly toxic Aβ oligomers and converts these into non-toxic, non-amyloidogenic and ThT-negative amorphous aggregates. In animal models, simple oral administration of D3 with drinking water achieves a situation wherein treated transgenic AD mice exhibit considerably fewer plaques and have significantly improved cognitive capabilities.
According to the prior art, Aβ oligomers are detected and quantified for example in an Aβ-containing, non-separated sample by means of oligomer-specific antibodies. This method is only semi-quantitative because, for each sample to be determined, a comparison standard is required which necessarily must be simultaneously entrained in the assay with the sample to be measured. Furthermore, this method is not reliable because the oligomer-specific antibodies possibly do not recognize all types of oligomer or do not recognize them to the same extent.
Furthermore, an Aβ-containing sample can be fractionated with the aid of different centrifugation techniques, so that different Aβ species are present in different fractions. These can then be analyzed by means of ELISA, Western Blot or SDS-PAGE, as is known for example from Funke et al. (S. A. Funke, T. van Groen, I. Kadish, D. Bartnik, L. Nagel-Steger, O. Brener, T. Sehl, R. Batra-Safferling, C. Moriscot, G. Schoehn, A. H. C. Horn, A. Muller-Schiffmann, C. Korth, H. Sticht, D. Willbold. Oral Treatment with the D-Enantiomeric Peptide D3 Improves the Pathology and Behavior of Alzheimer's Disease Transgenic Mice. ACS Chem. Neurosci. (2010), 1, 639-648).
A third method which is often used is the ThioflavinT (ThT) test, which, however, disadvantageously, only allows a reduction in the proportion of fibrils to be measured. According to what is presently known, this is not sufficient to identify a promising active ingredient candidate for oligomer reduction.
Disadvantageously, therefore, all the techniques based on antibody detection depend on the accessibility of the epitope. On account of different Aβ aggregate structures, however, the epitopes are sometimes hidden. SDS polyacrylamide gel electrophoresis (SDS-PAGE) analyses are independent of the problems mentioned above, but disadvantageously have a lower sensitivity and are not quantitative. SDS-PAGE is moreover disadvantageous since different bands of dimers, trimers and tetramers are formed and detected, since in particular the strongly aggregating samples form aggregates of high molecular weight. Said method is thus also disadvantageously only semi-quantitative because, for each sample to be determined, a comparison standard is required which necessarily must be simultaneously entrained in the assay with the sample to be measured.
The method for the quantitative characterization of amyloid and/or aggregating peptides and/or proteins in a sample comprises the following steps:                providing a sample, wherein the sample includes an amyloid and/or aggregating peptide and/or protein in at least one aggregate size and shape,        adding an active ingredient to be analyzed to the sample solution,        separating the amyloid and/or aggregating peptides and/or proteins from one another according to their aggregate size and shape. As a result, there is obtained from the sample a plurality of fractions which can be analyzed with regard to the concentration and in which the amyloid and/or aggregating peptides and/or proteins having a particular aggregate size and shape are present,        optionally completely denaturing the amyloid and/or aggregating peptides and/or proteins of a particular fraction into monomer building blocks,        determining the change in concentration of the peptide and/or protein building blocks by comparison against control values without the active ingredient.        
The problem is also solved by the following method.
The method for the quantitative characterization of amyloid and/or aggregating peptides and/or proteins in a sample comprises the following steps:                providing a sample, wherein the sample includes an amyloid and/or aggregating peptide and/or protein in at least one aggregate size and shape,        adding an active ingredient to be analyzed to the sample solution,        separating the amyloid and/or aggregating peptides and/or proteins from one another according to their aggregate size and shape to form multiple, at least 2, preferably at least 3, particularly preferably at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 fractions which can be analyzed. As a result, there is obtained from the sample a plurality of fractions which can be analyzed with regard to the concentration and in which the amyloid and/or aggregating peptides and/or proteins having a particular aggregate size and shape are present,        optionally completely denaturing the amyloid and/or aggregating peptides and/or proteins of a particular fraction into monomer building blocks,        determining the change in concentration of the peptide and/or protein building blocks in at least one fraction, preferably in at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 fractions, which can be analyzed with regard to the concentration, by comparison against control values without the active ingredient.        
Advantageously, therefore, any desired fraction can be analyzed by one of the abovementioned methods to ascertain changes in concentration after the active ingredient has been added. The method thus provides a method step which makes it possible to analyze more than one fraction for changes in concentration or for changes in aggregate size or other parameters. By virtue of the method, therefore, fractions are obtained which can be analyzed both quantitatively and qualitatively, and not only in respect of changes in concentration. In the context of the method, the term “desired fraction” encompasses in particular, but not exclusively, those fractions which, prior to separation, also contained aggregating and/or aggregated peptides and/or protein building blocks, in particular toxic oligomers.